PROJECT SUMMARY To better understand the role of histone variants and their post-translational modifications on the development, progression and treatment of AML and CLL, we intend to develop and apply high mass accuracy mass spec- trometry based assays to profile AML and CLL-specific patterns of histone isoforms and conduct detailed molecular characterization to identify the specific histone isoforms present in different genetic subtypes of AML and CLL and their impact on patient treatment and outcome. We will carry out these goals by profiling the state of histone modifications and characterize the changes in histone modifications that are induced by treatment with chromatin remodeling reagents and changes that are associated with specific genetic mutations. Specifi- cally we will focus on processes that correlate with induction of apoptosis and differentiation in vitro and in vivo in AML and CLL cells. These findings will be applied to patient tumor samples obtained from subjects under- going treatment with epigenetic therapies that target chromatin. The rationale is to define the changes in chromatin induced by these therapies and determine chromatin's potential to predict risk and outcome for the therapy. The techniques developed and validated in this proposal will allow basic scientists to detect and understand specific changes observed in histone isoforms and clinical-translational scientists to effectively predict outcome and apply therapies that modify these in patients with AML and CLL. The aims of this applica- tion are: Aim 1) To combine multidimensional liquid chromatography with high mass accuracy mass spectrometry to profile histones in malignant hematopoietic tumor cells. The technique will be applied to profile malignant cells from genetic subtypes of AML and CLL. We hypothesize this technique will facilitate the comprehensive characterization of histone variants and post-translational modifications and permit the target- ing of specific AML and CLL-specific sites not identifiable with currently available immunological techniques. Aim 2) To develop high mass accuracy tandem mass spectrometry assays to identify, map and characterize histone post-translational modifications in malignant cells from different AML and CLL genetic subtypes by high mass accuracy peptide mass mapping and liquid chromatography tandem mass spectrometry. We hypothesize these methods will allow the identification of select histone modifications associated with specific genetic subtypes of AML and CLL that are associated with poor clinical outcome and/or alterations due epigenetic therapy. Aim 3) To develop both: 1) well validated and generalizable analytical "pipelines", and 2) open-source software platforms that facilitate such "pipelines";which collectively facilitate analysis of high mass accuracy LC-MS and LC-MS/MS data obtained on histones. We hypothesize that improvement in informatics that support high-mass measurement accuracy measurements will significantly improve the characterization of histones as well as other proteins.